| Background:Acute myeloid leukemia (AML) is the most common hematological malignancy in ad μ Lts, characterized by distorted proliferation and development of myeloid cells and precursors in blood and bone marrow (BM). Even though Chemotherapy, the main therapy of AML, has made a great progress, the effect is still not ideal. The immune dysfunction of AML patients is one of the reasons which cause refractoriness and high mortality. T cell immune is an important part in anti-tumor immunity and its abnormal reg μ Lation network is the key factor to the pathogenesis and prognosis of AML. What’s more, T-helper (Th) cells, situated in a pivotal status in the immune system network, are the main reg μ Lators and effectors in the immune reaction. The past researches about Th cells were mainly confined to Thl and Th2cells in the peripheral blood of AML patients. Recently, Th17, T regμLatory cells (Tregs), Th22and Th9have been defined as four more novel distinct CD4+T subsets from Thl and Th2cells. It was broadly approved that the significantly decreased ratio of Thl/Th2and increased Treg cells percentage were existed in the peripheral blood of AML patients. There are several research rEports about Th17cells in AML patients, but there is a divergence. For Th22and Th9subsets, there is no rEport about their roles in solid tumor or AML. Moreover, the precise roles of the above Th subsets in BM microenvironment of AML patients are still not clarified.Objective:The purpose of the current study was to illustrate the specific constitution and immune regulation mechanisms of Th subsets (especially the novel recognized Th17/Treg, Th9and Th22subsets) in BM environment of AML patients and to explore their roles in AML pathogenesis, progress and prognosis. Our study may offer a novel therapeutic target for treatment of AML.Methods:We selected48newly-diagnosed (ND),34complete remission (CR) and19relapsed-refractory patients with AML enrolled from January2012to May2013in dEpartment of Hematology, Qilu Hospital, Shandong University and slight iron deficiency anemia patients as controls, and BM was collected from all the subjects, we examined the percentage of Thl, Th2, Th17, Treg, Th22and Th9cells and the alteration of Thl/Th2, Thl7/Treg ratio in each group by flow cytometry; the mRNA expressions of key transcription factor RORC, T-bet, GATA-3, AHR and Foxp3were investigated by RT-PCR. BM supernatants were harvested for detection of Thl-related cytokine (IFN-y), Th2-related cytokine (IL-4), Th17-related cytokine (IL-17), Treg-related cytokines (IL-10, TGF-β), Th22-related cytokine (IL-22), Th9-related cytokine (IL-9) and IL-6levels in each group by Enzyme linked immunosorbent assay (ELISA).ResμLts:1. Th22cells in BM of AML:Compared with control or CR AML patients, the percentage of Th22cells in ND or relapsed-refractory AML patients was significantly decreased (p<0.05). The level of IL-22in BM supernatants of ND or CR patients was markedly decreased compared with the level in controls (p<0.05). No significant difference of BM IL-22level between the three AML groups was observed (p>0.05).2. Th17cells, Tregs and their balance in BM of AML:2.1Th17frequency was markedly decreased in ND patients compared with CR, relapsed-refractory AML patients or controls (p<0.05). Although the BM plasma IL-17level in ND patients showed the downward trend, no statistical difference was found between these four groups (p>0.05). For the mRNA expression of RORC, the key transcription factor of Th17cells, the quantity was markedly increased in the three AML groups compared with controls (p<0.05).2.2Treg frequency was significantly increased in ND or relapsed-refractory patients compared to controls (p<0.05). Consistent with the flow cytometric res u Lts, we also found a significant increase of BM plasma IL-10level in ND patients compared with other three groups (p<0.05). TGF-P level was markedly decreased in ND and relapsed-refractory AML patients compared with the level in controls or CR patients (p<0.05). Foxp3mRNA expression was significantly elevated in ND or relapsed-refractory patients compared with in controls (p<0.05).2.3The distinct ratio imbalance of Th17/Treg was found in AML patients. Th17/Treg ratio was significantly decreased in ND or relapsed-refractory patients compared with the ratio in CR or controls (p<0.05).3. Thl, Th2and their balance in BM of AML:3.1The frequency of Thl cells in ND patients was lower than that in other three groups (p<0.05). Compared with CR group, Thl percentage was significantly decreased in relapsed-refractory group (p<0.05). Consistently, a significant decrease of plasma IFN-y level in ND and relapsed-refractory patients compared with the level in CR group was observed (p<0.05).3.2For Th2cells, significant decrease was found in ND compared with CR patients or controls (p<0.05). Correspondently, compared with controls, a significant increase of BM plasma IL-4level in ND patients was found (p<0.05). The mRNA expression of GATA-3, key transcription factor of Th2, was significantly deceased in three AML patients groups compared with in controls (p<0.05).3.3The distinct ratio imbalance of Th1/Th2was found in AML patients. Th1/Th2ratio was markedly decreased in ND group compared to CR group (p<0.05).4. Th9subset in BM of AML:For Th9subset frequency, no statistical difference was found between these four groups (P>0.05). However, we observed a statistical increase of plasma IL-9level in ND patients compared to the controls (p<0.05).5. Clinical relevance of BM Th subsets in AML patients:In ND AML patients, a positive correlation was found between Thl and Th17cells frequency (p<0.05). A significantly negative correlation was found between Thl cells frequency or Th2cells frequency and peripheral white blood cell (WBC) counts (p<0.05). Moreover, there is a significantly negative correlation between Thl7/Treg ratio or Thl/Th2ratio and BM leukemic blast (p<0.05).6. Chemotherapy partly ameliorated imbalanced Th subsets and related cytokines in BM microenvironment of AML:CR was obtained after the standard induction chemotherapy. The percentages of Thl, Th2, Th17and Th22cells were increased while Treg cells percentage was decreased when the AML patients achieved CR after chemotherapy (p<0.05). For the cytokines levels in BM supernatants, the levels of IL-10and IL-9were markedly decreased while TGF-β level was significantly increased after the patients got CR (p<0.05).7. The dynamic Bayesian network of Th subsets variation in AML patients showed that Th17subset was situated in a pivotal status of the network.Conclusions:1. The down-regμ Lation of Th22, Th17, Thl and their secreted cytokines, the up-reg μ Lation of Treg and immunoinhibitory cytokines, the shift ratio of Th17/Treg and Thl/Th2, and the aberrantly-expressed transcription factors may synergistically contribute to the suppressive immune system in AML BM.2. The extensive dynamic Bayesian network of Th subsets variation in AML BM showed that Th17subset was located in a pivotal status of the network and its balance with Treg subset affected directly or indirectly many other cytokines and mRNA expressions of the key transcription factors. Th17and Treg subsets were the key nodes in AML and may be the future research and intervention target of AML. |
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